Project Summary: Understanding how pluripotency is achieved and maintained is a critical first step in enabling researchers to induce pluripotency when it is needed or prevent it when it is harmful in cancer. One potential avenue of research lies in studying germ granules. Germ granules are heterogeneous ribonucleoprotein (RNP) complexes found in germ cell cytoplasm from worms to humans. In C.elegans, germ granules, known as P granules, are critical for maintaining fertility and germ cell pluripotency. Research done in the Updike lab has shown that depletion of P granules through RNAi triggers sterility and causes misexpression of sperm, muscle and pan- neuronal markers in the germ cells. This could be due to P granules antagonizing the accumulation of misexpressed transcripts or selectively repressing their translation. Distinguishing between these possibilities is critical to elucidating the function of P granules in pluripotency. However, research so far has been hampered by the technical limitations of P-granule RNAi. P granules are maternally inherited, thus RNAi must be initiated in the generation before analysis. Therefore, it is currently difficult to elucidate immediate and long term effects of induced P-granule loss. The work proposed in this application takes advantage of a new technique to deplete P granules called the auxin-inducible degradation (AID) system. P granules have three critical protein components ? PGL-1, PGL-2 and PGL-3. To degrade these targets CRISPR was used to engineer a degron tag to the end of all three endogenous genes in C. elegans. Then, upon auxin exposure, these worms (referenced as PgAID) will degrade PGL-1, PGL-2 and PGL-3 resulting in elimination of these nucleators of P-granule assembly and dispersal of P granules. These PgAID worms will allow for the identification of the immediate, mid-, and long-term effects of P- granule dispersal in the adult germline. This will be done by comparing PgAID worms to controls to identify alterations in germ-cell proliferation, sterility, brood size, and morphology due to P-granule dispersal. mRNA and reporter expression of select sperm and neuronal genes will also be examined and compared to previous data generated with the P-granule RNAi approach. PgAID worms will also be used to determine the sequential changes in mRNA expression following P-granule depletion. This will be done by performing mRNA-seq on dissected germlines immediately after P-granule dispersal, and at times before and at the onset of somatic reporter misexpression. Overall, this proposal will provide a critical next step in elucidating the mechanisms used in the germline to maintain cellular pluripotency, which will help unlock the possibility of successfully using these mechanisms for therapeutic benefit.